Simplifying Autoimmune Testing

As much as 20 percent of the world's population is afflicted with some form of autoimmune disease. Women represent 75 percent of cases, although it is not normally regarded as a women's health issue. There are approximately 80 known autoimmune diseases, which collectively affect every organ in the body. To support laboratory testing for autoimmune disease and to simplify the testing protocol, DPC has developed three assays: the automated ANA Screen* and dsDNA Antibody† assays; and the ANA lineBlot‡, an immunoblot for identifying 14 autoantibodies in a single test.

Autoimmunity is a general term to describe an immune response generated by the body against its own cells or tissues. Such a response is appropriate when the body directs immune processes against damaged or diseased cells. Detrimental or abnormal autoimmunity, however, represents a failure to tolerate autoantigens of normal cells. The general mechanisms of harmful autoimmunity include interaction of antibodies with cell-surface components, formation of autoantigen-autoantibody complexes in fluids with or without deposition in tissue, and sensitization of T cells.

Major systemic rheumatic diseases include
	Systemic lupus erythematosus (SLE)
	Drug-induced SLE
	Sjögren's syndrome
	Rheumatoid arthritis
	Scleroderma
	Polymyositis/dermatomyositis
	CREST syndrome
	Mixed connective tissue disease (MCTD).

ANA testing
Antinuclear antibodies (ANA) are antibodies directed against components of the cell nucleus, such as nucleoproteins and nucleic acids. These autoantibodies are associated with many systemic diseases, including SLE, Sjögren's syndrome, progressive systemic sclerosis (PSS), and MCTD. ANA tests may be used as diagnostic or prognostic indicators or as a means of monitoring therapy efficacy.

With nearly 100 percent of SLE patients testing positive for ANA by current ANA methods, SLE is the primary disease of focus for ANA screening. On the other hand, the ANA test is only one of eleven criteria used in the diagnosis of SLE. The specificity of most ANA methods allows for positive results in approximately 5 percent of the apparently healthy population. Diagnosis must therefore take the complete clinical picture into account.

Several techniques are used to screen for ANA: agglutination, indirect immunofluorescence (IFA), light microscopy, fluorescence immunoassay and enzyme immunoassay. IFA remains the predominant format for ANA screening, although enzyme immunoassays are becoming more widely accepted because of the subjectiveness of IFA interpretation.

A representative IFA protocol involves incubating diluted patient serum with a fixed substrate containing nuclei (Hep-2 cells or mouse liver). The slide is washed and then incubated with an anti-human immunoglobulin antibody conjugated with fluorescein. After a second wash, the slide is read under a fluorescence microscope; fluorescent staining of cellular components indicates a positive test. This staining appears as any of several patterns commonly identified as diffuse, peripheral, speckled, homogeneous, nucleolar, centromere, and spindle fiber.

The DPC ANA Screen, an automated enzyme immunoassay developed for use on the IMMULITE^® family of immunoassay systems, employs a solid phase (bead) coated with a combination of 14 antigens. The conjugate is an anti-human IgG antibody labeled with alkaline phosphatase. Results are obtained after approximately 60 minutes, using a chemiluminescent substrate for detection.

Traditional testing paradigm
In the traditional testing paradigm for autoimmune disease, patient serum is screened for ANA using either the IFA or EIA method. When the IFA method is positive, both titer and pattern are normally reported; for an EIA, a qualitative result is reported. In many cases, positive results are followed up with an IFA titer and pattern. After the ANA screen, the physician may order additional testing based on the clinical picture. This may consist of a quantitative dsDNA antibody assay, if SLE is a likely diagnosis, or additional testing for antibodies to extractable nuclear antigens (ENA). These ENAs typically include SSA (Ro), SSB (La), Sm, RNP, Scl-70 and Jo-1. These antigens can be tested individually using a variety of methods such as EIA or Ouchterlony plates, or they can be included with 4 to 6 different ENAs and utilized as an additional screening tool. In some cases it may be difficult for the physician to determine which ENA to order, making a third round of testing necessary.

New ANA testing paradigm
DPC's qualitative ANA Screen and quantitative dsDNA Antibody assays will soon be available on all of the automated IMMULITE platforms. (DPC continues to offer the isotopic Anti-DNA [Farr] assay as well.) In addition, DPC offers the ANA lineBlot which allows profiling and identifying of 14 separate autoantibodies at once, minimizing the need for additional testing. These tests allow for a simplified testing paradigm that uses the concept of Screen, Profile and Quantify (Figure 1).

DPC assay features
The ANA Screen is a qualitative two-cycle assay with a 1 to 40 predilution. Results are available in about an hour. The antigens coated on the bead include dsDNA, SSA, SSB, Sm, RNP, Scl-70, Jo-1, histone, centromere and ribosomal RNP. The Hep-2 nuclear extracts are spiked with recombinant antigens resulting in better clinical sensitivity and specificity, a necessary feature for accurate rheumatic disease autoantibody testing.

The dsDNA Antibody assay is a quantitative two-cycle assay with a 1 to 20 predilution. It, too, provides results in about an hour. The assay measures anti-dsDNA IgG with minimal crossreactivity to single-stranded DNA, for better specificity. Assay results, obtained for clinically well-defined active SLE patients and apparently healthy individuals at two sites, demonstrated a clinical sensitivity of 97% for active SLE patients and a clinical specificity of 99.2% for apparently healthy subjects.1

ANA lineBlot is a simple line blot test that identifies 14 autoantibodies on a single nitrocellulose strip in roughly 2 hours. Highly purified, specific antigens are applied to the strip, providing an easy-to-read and highly specific profiling assay. Antigens include dsDNA, SSA, SSB, Sm, RNP, Scl-70, Jo-1, histone, centromere and ribosomal RNP. The assay can be run manually or using an automated blot processor. Included on each blot strip is a reagent control to verify addition of sample, and a cutoff control for distinguishing between positive and negative results for individual antinuclear antibodies.

With DPC's expanding autoimmune product line, the clinical laboratory will have an effective automated means of performing autoimmune testing, providing physicians with a complete systemic rheumatic disease profile in three easy steps.